Almost all bacterial pathogens encode proteins that directly misregulate host cells. Most notable are Gramnegative organisms encoding specialized secretion systems that translocate substrates into host cells. Some of these organisms translocate as many as 200 different proteins into host cells. Attempting to understand how this menagerie of proteins is coordinated is difficult, as these proteins often have activities that appear to antagonize each other when studied in isolation. It is unlikely, however, that these proteins act at cross-purposes, and instead they presumably collaborate to promote the desired effects on host cells. If the consequences of collaboration can be identified, then this information can be used to identify potential sites for therapeutic intervention in the disease process, as it is likely that this approach will lead to the identification of a limited number of targets in the host controlled by a large number of bacterial proteins. This is based on the idea that groups of proteins with very different functions work together to produce a single effect in the host cell. This proposal intends to identify how bacterial virulence factors collaborate, focusing on proteins translocated via the Type III Secretion System (TTSS) of Yersinia pseudotuberculosis. In particular, interactions between three bacterial components will be dissected: invasin, the TTSS and the translocated proteins YopE and YopT. Invasin and the Yops appear to antagonize each other. Invasin promotes bacterial entry into host cells resulting from integrin engagement and activation of small GTPases, while these Yops interfere with uptake, by misregulating small GTPases. This application will test the hypothesis that, rather than compete with each other, these proteins collaborate to allow misregulation of host cells. Experiments are proposed to determine if small GTPase activation by invasin brings the targets of the Yops into close proximity to bacteria that are bound to host cells, or if it is primarily directed at stimulating translocation of Yops. To investigate these models, strategies will be developed that allow the tracking of small GTPases originating from several sites in the cell, following their routes as they encounter YopT after bacterial binding. In the process, the site of action in the host cell of Yops that inactivate small GTPases will be identified. This would be the first demonstration that a bacterial adhesion protein and translocated substrates work together to misregulate small GTPases. The ability to disrupt collaborative bacterial virulence factor interactions that promote the disease process is critical for identifying strategies that maintain immune function in the presence of pathogen attack.